stromal phosphateconcentration is low during feedback ... · stromal phosphateconcentration is...

Plant Physiol. (1989) 91, 679-6840032-0889/89/91 /0679/06/$01 .00/0

Received for publication February 27, 1989and in revised form May 1, 1989

Stromal Phosphate Concentration Is Low during FeedbackLimited Photosynthesis1

Thomas D. Sharkey* and Peter J. VanderveerDepartment of Botany, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

It has been hypothesized that photosynthesis can be feedbacklimited when the phosphate concentration cannot be both lowenough to allow starch and sucrose synthesis at the requiredrate and high enough for ATP synthesis at the required rate. Wehave measured the concentration of phosphate in the stroma andcytosol of leaves held under feedback conditions. We used non-aqueous fractionation techniques with freeze-clamped leaves ofPhaseolus vulgaris plants grown on reduced phosphate nutrition.Feedback was induced by holding leaves in low 02 or high CO2partial pressure. We found 7 millimolar phosphate in the stromaof leaves in normal oxygen but just 2.7 millimolar phosphate inleaves held in low oxygen. Because 1 to 2 millimolar phosphatein the stroma may be metabolically inactive, we estimate that inlow oxygen, the metabolically active pool of phosphate is be-tween negligible and 1.7 millimolar. We conclude that halfwaybetween these extremes, 0.85 millimolar is a good estimate ofthe phosphate concentration in the stroma of feedback-limitedleaves and that the true concentration could be even lower. Thestromal phosphate concentration was also low when leaves wereheld in high C02, which also induces feedback-limited photosyn-thesis, indicating that the effect is related to feedback limitation,not to low oxygen per se. We conclude that the concentration ofphosphate in the stroma is usually in excess and that it issequestered to regulate photosynthesis, especially starch syn-thesis. The capacity for this regulation is limited by the couplingfactor requirement for phosphate.

During photosynthesis, phosphate is required by the cou-pling factor for the production of ATP from ADP. At thesame time, phosphate inhibits starch (15) and sucrose (32)synthesis, as well as many of the reactions of the carbonreduction cycle (19, 24). Sharkey (25) suggested that theconflicting requirements of ATP synthesis for phosphate andstarch and sucrose synthesis for low phosphate can limit theoverall rate of photosynthesis at high rates of photosynthesis.This condition is called the feedback limitation of photosyn-thesis (26). Because the affinity of the coupling factor forphosphate is high (1, 16, 23) and the sensitivity of starch andsucrose synthesis to phosphate is also high, it was predictedthat the concentration of phosphate in the stroma would fallto 1 mM, perhaps even less, during feedback-limited photo-synthesis (25). Furbank et al. (6) suggested that RuBP carbox-ylase reduces the affinity of the coupling factor for phosphate

Research supported by U.S. Department of Energy grants DE-FG02-87ER60568 and DE-FG02-87ER 13785.

and so the phosphate level need not fall to such low levels tolimit photosynthesis.The concentration of phosphate in the stroma has been

measured in the past (3, 21, 36), but it has never beenmeasured in feedback-limited leaves, that is leaves whichexhibit 02-insensitive photosynthesis. We decided to measurethe concentration of phosphate in the stroma and cytosol ofleaves exhibiting feedback-limited photosynthesis. This con-dition can be induced by feeding the phosphate sequesteringagent mannose (9) and can also occur under natural condi-tions ( 13, 20).The measurement of stromal phosphate concentration is

difficult because plants grown on luxuriant levels of phos-phate, as is common practice in research, usually have a largeamount of metabolically inactive phosphate in the vacuole(4, 39). This problem can be overcome by growing plants withmore realistic phosphate nutrition. When the phosphate sup-ply is restricted, the phosphate concentration in the vacuolecan be substantially reduced with little or no effect on pho-tosynthesis (5, 22). Rebielle et al. (17) found that sycamorecells starved for phosphate lose primarily vacuolar phosphate,preserving the concentration of phosphate in the cytoplasm.This observation justifies the study of stromal phosphateconcentration in plants grown on restricted phosphate nutri-tion, especially if it can be shown that such plants have normalstromal phosphate concentrations under some conditions. Weused plants fertilized with Hoagland solution modified tocontain 15% of the usual amount of phosphate.

Phosphate is extremely soluble in water. Therefore, frac-tionation of plant material for compartmental analysis ofphosphate must be done nonaqueously. We chose the methoddeveloped by Gerhardt and Heldt (8).We report here measurements which confirm the prediction

that the concentration of metabolically active phosphate inthe stroma can fall to 1 mm or less. We measured the concen-tration of phosphate in the stroma and cytosol of feedbacklimited leaves.

MATERIALS AND METHODS

Plant Culture

Plants of Phaseolus vulgaris L. cv Linden were grown in agrowth chamber in 4 L pots containing a soil:peat:perlite:ricehull (3:3:3:2) mix. Plants were grown under a 12 h photoper-iod with 24/17°C day/night temperature, 60% RH with aphoton flux density of 500 gmol m-2 s-'. The plants werefertilized 5 time per week with Hoagland's solution B (10)

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SHARKEY AND VANDERVEER

modified to contain just 15% of the normal concentration ofphosphate (0.15 mM Pi).

Gas Exchange and Leaf Sampling

Gas exchange measurements were carried out in an opengas exchange system as described in (34). Air was mixed fromN2, 02, and CO2 so that the partial pressures of 02 and CO2could be controlled. Once the gas exchange characteristics ofeach leaf were determined, the leaf was quickly frozen in afreeze-clamp apparatus. We estimate that fewer than 250 mselapsed between the time the light illuminating the chamberwas interrupted and the time the temperature of the leafmaterial was less than 0°C. Six leaf samples were combinedto make one sample for nonaqueous fractionation. Samplesfor different treatments within an experiment were taken fromopposite leaflets of trifoliolates for uniformity. Samples werestored at -80°C for up to 1 week.

Nonaqueous Fractionation

Nonaqueous fractionation of the leaf material was carriedout using methods similar to those ofGerhardt and Heldt (8).The frozen leaf samples were ground in liquid N2 in a mortar.The leaf powder was lyophilized in a Virtis 10-324 until thepressure was 5 mtorr. This usually required about 3 h. Wemade comparisons between this method which allows thesample to warm up once the water is lost and the slowermethod of holding the sample at -40°C during the freeze-drying. The latter method requires three days for completedrying, and we found the results to be indistinguishable. Thedry powder was transferred to 20 mL heptane and sonicatedwith a Branson Sonifier 250 on power setting 8 and 50% dutycycle. The tube containing the leaf powder suspended inheptane was held in a beaker containing heptane kept partiallyfrozen by the addition of liquid N2. The suspension wasconcentrated by centrifugation then put on a 7 mL discontin-uous density gradient ofheptane and tetrachloroethylene. Thegradient was made by putting one ml of the following densi-ties: 1.60, 1.55, 1.50, 1.475, 1.45, 1.40, and 1.35. Thegradientwas centrifuged at 25,000 g in an HB-4 swinging bucket rotorin an RC-SB refrigerated centrifuge for 2.5 h. After centrifu-gation, the gradient was divided into six 1 to 2 mL fractions.Each fraction was divided with one third of the fraction forenzyme assays and two-thirds for metabolite assays. Heptanewas added to each sample, then each sample was centrifugedin a microcentrifuge for 4 min. The pellet was allowed to dryovernight under reduced pressure in a desiccator containingparaffin and silica gel.

Marker Enzyme Assays

For enzyme assays, the samples were resuspended in 500IAL 100 mm Bicine (pH 7.8), 5 mM MgCl2, and 1 mm EDTA.The samples were sonicated for 30 s. After 5 min, the sampleswere centrifuged for 5 min in a microcentrifuge. The super-natant was used for enzyme assays and the pellet was used forChl determinations. All manipulations were carried out onice.Chl was used for the chloroplast marker. In many experi-

ments we also measured NADP-dependent glyceraldehyde 3-phosphate dehydrogenase and found no significant differencesin distribution of these two chloroplast markers in the densitygradient. Chl was determined by adding 1 mL 95% ethanolto the pellet, sonicating for 30 s, then centrifugating for 2 minin a microcentrifuge. TheA at 654 nm was read and convertedto Chl amount using the equations in Wintermans andDeMots (37).PEPCase2 was used as the cytosol marker. PEPCase was

assayed at 22°C by adding 10 to 30 ,l of extract to 250 AL100 mm Bicine (pH 7.8), 5 mM MgCl2, 1 mM EDTA, 550 AMNADH, 15 units malate dehydrogenase, 4 mM NaHCO3, with2.5 Ci mol' NaH'4CO3. The reaction was started by addingPEP to give a final concentration of 2 mm. After 5 min, thereaction was stopped by adding 500 ,ul 2 N HCI. The assayswere dried overnight. The next morning, 100 ,uL of water and3 mL of Bio-Safe II scintillation cocktail was added to eachsample. After vortexing, the samples were counted in a scin-tillation counter.

For the vacuolar marker we used a-mannosidase. Thisenzyme was assayed by adding 10 to 50 ,uL extract to 400 ,uL50 mm citrate (pH 4.5) and 400 ,uL 5 mm p-nitrophenyl a-D-mannoside. After 30 min at 37°C, the assay was stopped byadding 400 ,uL 0.8 M borate (pH 9.8). The A at 405 nm wasread.

Inorganic phosphate was measured after extraction of themetabolite fraction in 600 ,L 3.5% perchloric acid. Theextracts were neutralized to pH 6 to 7 by adding a solution of2 N KOH, 150 mm Hepes (to help stabilize the pH), and 10mM KCI (to help the precipitation of KC104). The phosphateassay was the malachite green enhanced-molybdate assaytaken from Itaya and Ui (11) and Penny (14). An assaysolution of 2 g L' malachite green (Sigma M9636) and 10mM ammonium molybdate in 0.8 M HCI was made up atleast two days prior to assay. This solution was filtered throughWhatman No. 1 filter paper. Plant extract (10-50 ,uL) wasadded to 800 ,uL of molybdate reagent. After 1 min, 100 ,uL1 M trisodium citrate was added to the assay. After 1 furthermin, 100 ,uL of 1% Extran 1000 detergent was added to theassay. The optical density at 650 nm was read after 30 minand compared with standards made with dried KH2PO4.

Data Analysis

In principle it is possible to set up simultaneous equationsto solve for the phosphate concentration in each of the threecompartments, stroma, cytosol, and vacuole (8). In practice,we found that a two-compartment analysis as described byGerhardt and Heldt (8) and as we describe below gave moreconsistent and believable results. The two-compartmentanalysis is as follows (8). The amount of phosphate in thestroma, P., is related to the amount of stromal marker. Weused Chl as the stromal marker. The amount of phosphate in

2Abbreviations: PEPCase, phosphoenolpyruvate carboxylase; PEP,phosphenolpyruvate; RuBP, ribulose bisphosphate; PGA, 3-phos-phoglyceric acid.

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STROMAL PHOSPHATE CONCENTRATION

the cytosol is related to the amount of the cytosolic markerPEPCase.

pstr

a Chl (1)

pc,>t

PEPCase (2)

In each fraction, denoted by a subscript i, we assume that thetotal phosphate present comes only from the stroma andcytosol.

p total = p,str + pIcy (3)

Substitute Equations 1 and 2 into Equation 3

pptotal = a Chli + b PEPCase; (4)

Divide by PEPCasep total Chl

PEPCase PEPCase +b (5)

Given Equation 5, if we plot P1/PEPCasej versus Chl1/PEP-Casei, the slope is a, the ratio of stromal phosphate to Chl andthe y intercept is the ratio of cytosolic phosphate to PEPcarboxylase. By multiplying the y intercept by the ratio ofPEP carboxylase to Chl, the ratio of cytosolic phosphate toChl is obtained.

Because this analysis assumes that phosphate in each frac-tion is derived only from the stroma or the cytosol, it isnecessary to estimate the contribution of the vacuole to de-termine the error of this analysis. The distribution of markersfor one of the gradients is shown in Figure 1. Over 70% ofthe vacuolar marker was in the heaviest two fractions. Thetop four fractions contained less than 10% each of the vacu-olar marker. We used only the top three or four fractions inthe analyses reported in the paper so that vacuolar contami-nation was kept to a minimum. To estimate the error intro-duced by this contamination, we assumed that all of thephosphate in the heaviest fraction was vacuolar (an overesti-mation). The ratio ofphosphate to vacuolar marker calculated

00

-4-i

0a)

E

'4-

cC

0

L-

a-

= Chi0.5,

IPEPCase _ a-mannosidase

0.4

0.3

0.2

0.1

0.

0 1 2 3 4 5Light Density gradient fraction

6Heavy

Figure 1. Distribution of markers in the fractions of a density gradientof freeze-dried plant material. Each marker is given as a proportionof the total amount of that marker in the entire gradient.

this way was then applied to the top fractions. This analysisindicated that less than 10% of the phosphate in the topfractions came from vacuolar contamination. If the ratio ofvacuolar content to cytosolic content remained constant overthe top four fractions then the cytosolic phosphate contentmay be overestimated by 10%. Because we were looking forlow phosphate levels, and ignoring the vacuolar phosphatewas likely to cause an overestimation of the phosphate con-centration in the stroma and the cytosol, we believe that it isa conservative and justifiable approach to ignore the vacuolarphosphate.

Chloroplasts from leaves which had been freeze-clamped inlow P(02) had a greater density, so we often could only usethe top three fractions of the gradient in the analysis insteadof the top four as for normal P(02) samples. Although theconcentration of phosphate in the stroma and cytosol can bedetermined from just two points, using three or four pointsserves as a check on the internal consistency of the data.Therefore, we report the number of points used and thecorrelation coefficient of the linear regression (r) for eachmeasurement reported.

RESULTS

Leaves freeze-clamped in low p(02) had much less stromalphosphate than leaves clamped in normal p(02) (Fig. 2). Whileonly two data points are required for the determination of theslope, using up to four data points averages out experimentalerror to give a more reliable estimate ofthe stromal phosphateconcentration. The fact that all four points approximate a

straight line is an indication of the internal consistency of themeasurement. Chloroplast material from leaves freeze-clamped in low P(02) was heavier than that from leaves freeze-clamped in normal P(02), and so the greatest ratio of Chl to

5--

E

X4-@Ec 3-- Normal 02

1 G-LowO

Chl/ PEPCase, Ag/mUFigure 2. Plot of Pi/PEP carboxylase versus Chl/PEP carboxylaseas described in the "Materials and Methods." The normal oxygenlevel is 200 mbar and the low level is 20 mbar. The open trianglesare from fractions 5 and 6 of the low oxygen sample and are notincluded in the calculations. The filled triangles are for fractions 5 and6 of the normal oxygen sample. The slope of the normal oxygen line(excluding fractions 5 and 6) is 0.248, and so there is 248 nmolphosphate/mg Chl in the stroma, whereas the slope of the low oxygenline (excluding fractions 5 and 6) is 0.053, indicating 53 nmol phos-phate/mg Chl in the stroma of the leaves frozen in low oxygen. Thecorrelation coefficients were 0.97 for the normal oxygen treatmentand 0.96 for the low oxygen treatment.

681




PEP carboxylase was less in the low compared to that in thenormal P(02) sample. We do not know why chloroplasts fromfeedback limited leaves were denser but speculate that it wasrelated to the differences in metabolite contents.The measurement of phosphate was repeated three other

times over a period of 6 months. The degree of phosphatestarvation of the plants varied from one experiment to thenext and so the rate of photosynthesis was different in thethree experiments (Table I). The variation in the degree ofphosphate starvation was caused by variations in age of theplants and in phosphate binding capacity of the soil used (andprobably other factors as well). Nevertheless, within eachexperiment, no stimulation of photosynthesis was found uponswitching to low P(02). The rate ofphotosynthesis varied fromwell below normal (experiment 1) to essentially normal (ex-periment 3).

Although the plants used for each measurement were phys-iologically different, the results were similar enough to justifycalculation of an average for all of the measurements that wemade (values in Table I and Fig. 2). These averages arereported in Figure 3. It is readily apparent that leaves held inlow P(02), under conditions where low p(02) does not stim-ulate photosynthesis, had a lower stromal concentration ofphosphate but similar cytosolic concentration of phosphatecompared to leaves in normal p(02)-

It is believed that the effect oflow oxygen on photosynthesisresults primarily from feedback limitations in photosynthesis(25, 26). Therefore, many of the effects caused by low oxygenshould also be observed in high p(CO2). We measured thephosphate concentrations in leaves held in normal or highp(CO2) (Table II). High CO2 caused the phosphate concentra-tion in the stroma fall, just as low 02 had.We tested the effect of feeding mannose on the concentra-

tion of phosphate in the stroma and cytosol. After feedingmannose, the concentration of phosphate in the stroma fell(Table II). Mannose-fed leaves had less cytosolic phosphate(Table II).The concentration of phosphate in the stroma and cytosol

of phosphate-starved plants was measured over a range ofphoton flux densities to characterize the phosphate-starvedplants (Fig. 4). The phosphate concentration in the stromawas relatively constant over a range of photon flux densities.

In the cytosol, the phosphate concentration was very high indarkness and at 100 ,umol m-3 s-'. At higher photon fluxdensities the cytosolic phosphate concentration fell.

DISCUSSION

The concentration of phosphate in the stroma falls whenleaves are put in low p(02). Assuming 25 ,uL mg-' Chl, theaverage phosphate concentration in the stroma of leaves heldin low p(02) was 2.7 mM; in two experiments the concentra-tion was just 2 mm and it was 2 mm in one of the measure-ments of leaves incubated in high CO. Some ofthis phosphatemay not be readily available for metabolism (38). The meta-bolically unavailable phosphate in the stroma was estimatedby Furbank et al. (7) by measuring the phosphate concentra-tion inside chloroplasts unable to photosynthesize for lack ofphosphate. They found between 1.5 and 2.5 mm phosphateinside such chloroplasts. Robinson and Giersch (18) found1.1 to 1.8 mm phosphate in phosphate-starved chloroplastswhen measured with a colorimetric assay, as employed in thisstudy. However, when they assayed the amount of phosphatethat readily exchanged with 32P-phosphate, they found only0.2 mm phosphate. Therefore, between 1 and 2 mm stromalphosphate measured colorimetrically must be considered met-abolically inactive. This amount could be bound to enzymesor inside membranes and released upon extraction. From thedata reported here and the data of Robinson and Giersch (18)and Furbank et al. (7), we conclude that the stromal phosphateconcentration in leaves in low P(02) may be anywhere fromvanishingly small (the 2 mM measured in the experimentreported in Figure 2 and experiment 2 of Table I, minus 2mM metabolically inactive phosphate) up to 1.7 mm (theaverage reported in Fig. 3 minus 1 mm metabolically inactive).In the absence of other compelling evidence our best guess isthat the concentration of metabolically active phosphate inthe stroma of feedback limited leaves is 0.85 mm and possiblylower.

In experiment 3, Table I, the phosphate concentration waslow, even at normal P(02). We interpret this result to indicatethat the leaves were feedback limited even in normal P(02) inthis experiment. The concentration of phosphate in thestroma of leaves in normal P(02) was usually in the range of

P(02) Stroma Cytosol

136163

CorrelationCoefficient

r (n)

Assimilation

imo/m-2s-1

0.94 (4) 8.5 ± 1.20.93 (3) 8.2 ± 1.2

240 222 0.98 (4) 11.9 ± 1.351 220 0.88 (3) 11.3 ± 2.2

89 194 0.94 (5) 18.3 ± 1.580 209 0.93 (3) 18.7 ± 0.7

'200| T

E 15000c 100Q)a

n2- 500

0

Stroma CytosolFigure 3. Average and standard error for all measurements ofstromal and cytosolic phosphate concentrations in leaves frozen innormal or low oxygen.

Table 1. Stromal and Cytosolic Phosphate Concentration andAssimilation Rates of Leaves in Normal or Low p(O2)

Leaf temperature was 24°C and C02 partial pressure was 600pbar.

nmol Pi mg-' Chl

14888

mbar

Experiment 120020

Experiment 220020

Experiment 320020




STROMAL PHOSPHATE CONCENTRATION

Table II. Stromal and Cytosolic Phosphate Concentration andAssimilation Rates of Leaves in Normal or High p(CO2) or afterFeeding Mannose

Leaf temperature was 240C and the 02 partial pressure was 200mbar.

p(C02) Stroma Cytosol Correlation AssimilationCoefficient

ubar nmol P mg-' Chl r (n) pmol m-2 s-'350 117 166 0.98(4) 16.1 ±2.01500 74 196 0.99 (4) 23.6 ± 1.2350 + mannose 78 110 0.98 (4) 13.8 ± 0.1

7 mM (Fig. 3). The stromal phosphate concentration was

similar over all photon flux densities. This concentration issimilar to the estimates of stromal phosphate concentrationpublished earlier (21, 36), but lower than other estimatesmade with chloroplasts with 15% vacuolar contamination (3).The decline in stromal phosphate concentration by 5 mM

upon switching to low P(02) under feedback conditions iseasily accounted for by the buildup of RuBP (27) and PGA(29) that occurs under these conditions. The increase in PGAconcentration is caused by a lowered ATP/ADP ratio (29)while the buildup of RuBP is caused by reduced carbamyla-tion of RuBP carboxylase (2, 28). The decline in carbamyla-tion is caused by the reduced ATP/ADP ratio (33).

Feeding mannose did not precisely mimic the naturallyoccurring feedback syndrome. Mannose sequestered phos-phate in the cytosol (Table II) as expected (9, 31, 35), andreduced hexose monophosphate and phosphoglycerate con-

centration (data not shown). Leaves fed a similar phosphatesequestering agent, 2-deoxyglucose, have much less RuBP(28). Feedback limited leaves have increased RuBP and phos-phoglycerate levels and constant or increased cytosolic phos-phate concentration (this occurred in three offour low oxygenexperiments and three of three high CO2 experiments).During feedback limited photosynthesis, the stromal phos-

phate concentration is extremely low (Fig. 3), yet the concen-

tration of RuBP is high (27). This condition makes no sense

when the feedback limitation is viewed as a lack of phosphateor inadequate phosphate nutrition (12, 30, 31, 35). However,if the low concentration of phosphate in the stroma is viewedas an intermediary, reporting that the capacity for triosephosphate production exceeds the capacity for triose phos-phate utilization, then the high concentration of RuBP isadaptive. Ordinarily, the concentration of phosphate insidethe stroma is saturating for ATP synthesis. As the rate ofphotosynthesis increases, starch and sucrose synthesis mustalso increase so that they are matched to the capacity fortriose phosphate production by the chloroplast. As light wasincreased up to 400 umol m-2 s-', the cytosolic phosphatelevel fell, but there was little decline in stromal phosphateconcentration (Fig. 4). Presumably P-glycerate increased tostimulate starch synthesis. Upon switching to low 02 or highCO2 to upset the balance between triose phosphate productionand consumption, the stromal phosphate concentration fallsdramatically providing a strong signal for starch synthesis.The decline in stromal phosphate can be effected by decar-

-1000-

800-

600E

Q; 400-

u3 200(0

0-0 100 200 300 400 500

Photon Flux Density, 1umol m-2 s1Figure 4. Stromal and cytosolic phosphate concentration versusphoton flux density. Leaves were freeze-clamped in the growthchamber, shade cloth was used to provide the different photon fluxdensities.

bamylation of RuBP carboxylase (2) leading to accumulationof RuBP at the expense of free phosphate.However, there is a limit to how far the stromal phosphate

concentration can fall before it will begin to limit ATP syn-thesis. Once this limit is reached, photosynthesis cannot in-crease further. A higher stromal phosphate concentrationwould inhibit starch synthesis while a lower concentrationwould inhibit ATP synthesis. Thus the high RuBP concentra-tion is adaptive in lowering the stromal phosphate concentra-tion to the point where starch synthesis is stimulated as muchas possible; in extremis ATP synthesis is inhibited and so

photosynthesis becomes feedback limited.The mechanism put forward above assumes that phosphate

is ordinarily in excess supply, and that changes in RuBPcarboxylase carbamylation sequesters more or less of theexcess as RuBP to regulate photosynthesis. This view is dif-ferent from the view put forward by Sivak and Walker (30,31) and Walker and Sivak (35). Their view, that phosphate isin short supply and easily becomes limiting in its own right,fails to explain the high concentration of RuBP that is foundin feedback limited leaves; our view explains that high con-

centration.

ACKNOWLEDGMENTS

Dr. Richard Gerhardt provided invaluable instruction in the non-

aqueous fractionation technique, and T. D. S. thanks Prof. HansHeldt for the opportunity to visit his laboratory to learn this techniqueand for discussions on the role of phosphate in photosynthesis.

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0 Cytosol

0

),-0 ~ Stroma0 -- 0~~

_

683




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stromal phosphateconcentration is low during feedback ... · stromal phosphateconcentration is...

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